1. Field of the Invention
The present invention relates to an enzyme-treated hesperidin with a superior water solubility, as well as to its production and uses.
2. Description of Prior art
Hesperidin is the name of a compound where, as seen in the formula I!, rutinose or L-rhamnosyl-(alpha-6)-glucose is bound via beta-linkage to the hydroxyl group at 7-position in hesperetin or 5,7,3'-trihydroxy-4'-methoxyflavanone. ##STR1##
Hesperidin, which is contained in unripe pericarps of citruses, is used in medicines and cosmetics as a source of vitamin P. Vitamin P exhibits a variety of physiological activities such as those of strengthening the capillary blood vessel, preventing bleeding and regulating blood pressure. Hesperidin is soluble in aqueous alkaline solution but substantially insoluble in water and acids: Fifty liters of water dissolves only about 1 g hesperidin (about 0.002 v/v %) at ambient temperature. For example, even when a small amount of hesperidin is contained, in the syrup of canned products, the syrup may become turbid and impair their commercial value.
A variety of methods have been proposed which may prevent turbidity in syrups due to the presence of hesperidin.
For example, Japanese Patent Kokai No. 7,593/91 discloses a process to produce an enzyme-treated hesperidin with an elevated water solubility, where a saccharide-transferring enzyme, in particular, an enzyme possessing alpha-glucosyltransferase activity is allowed to act on hesperidin in the presence of a partial starch hydrolyzate as alpha-glucosyl saccharide compound to form alpha-glucosyl hesperidin as represented by the formula II!. ##STR2##
Alpha-glucosyl hesperidin is, as seen in the formula II!, either a single compound where n (1 to 20) units of glucose (G) are sequentially bound via 1,4-linkage to the glucose at the 4-position in hesperidin as represented by the formula I!, or a mixture of alpha-glucosyl hesperidins which differ from each other in the number of the glucose units.
In the above mentioned enzymatic reaction, 40 to 80% hesperidin in material liquid is converted into alpha-glucosyl hesperidin during the enzyme treatment, while 20 to 60% hesperidin remains intact. Although intact hesperidin exhibits a relatively high solubility in aqueous solution when alpha-glucosyl hesperidin coexists, an elevated ratio of intact hesperidin to alpha-glucosyl hesperidin results in the insolubilization and precipitation of hesperidin within a short time period.
One may contemplate another method which may prevent the precipitation of intact hesperidin, where glue materials such as carboxymethyl cellulose are added to solutions with hesperidin to elevate their viscosity. Such method is, however, generally unacceptable because the addition of glue material may reduce customer interest, as well as because the use of glue material is prohibited in products to be exported.
There is available still another method which may retard the precipitation of intact hesperidin, where intact hesperidin is precipitated and then removed by separation using, for example, filtration, thus decreasing the ratio of intact hesperidin to alpha-glucosyl hesperidin.
This method, however, does not provide complete solutions because the intact hesperidin still causes precipitation after a lapse of prolonged time period.
There is available still another method, where fractions of alpha-glucosyl hesperidin are isolated from an aqueous solution containing both alpha-glucosyl hesperidin and intact hesperidin by means, for example, of chromatographic separation, prior to its use. This method is, however, unacceptable from an economical viewpoint because it may result in increased cost.
To solve these problems, the present inventors have the found that when alpha-glucosyl hesperidin and intact hesperidin in solution are subjected to alpha-L-rhamnosidase (E.C.3.2.1.40), the former alpha-glucosyl hesperidin undergoes no changes and substantially remains intact, while the latter hesperidin is hydrolyzed into rhamnose and monoglucosyl hesperetin as represented by the formula III!. This reaction yields an enzyme-treated hesperidin with a very superior water solubility which does not cause tubidity even when allowed to stand over a prolonged time period. The present invention is based on these findings.
The method, in which, in order to elevate the water solubility of hesperidin, alpha-L-rhamnosidase is allowed to act thereon to convert hesperidin into beta-monoglucosyl hesperetin, has been practiced on an industrial scale to prevent turbidity in the syrup of canned tangerines.
Prior Art, however, does not disclose the method of the present invention, where hesperidinase as an enzyme possessing alpha-L-rhamnosidase activity is allowed to act on a solution containing both alpha-glucosyl hesperidin and intact hesperidin so that the rhamnose moiety in the former alpha-glucosyl hesperidin is left intact, while that in the latter hesperidin is specifically hydrolyzed to change hesperidin into beta-monoglucosyl hesperetin, thus elevating the water solubility of the solution.
Japanese Patent Kokai No.80,177/96 discloses a method to prevent the precipitation of hesperidin crystals, where a solubilized hesperidin is added to a solution with hesperidin. Said Japanese Patent Kokai describes that the solubilized hesperidin is a compound where 1 to 10 or more glucose moieties are sequentially bound via alpha-1,4-linkage to the 4-position of the glucose moiety in hesperidin. This reference also teaches that the solubilized hesperidin can be produced by subjecting hesperidin in the presence of cyclodextrin to a saccharide-transferring enzyme which may be CGTase or 1,4-alpha-D-glucan; 1,4-alpha-D-(1,4-glucano)-transferase (E.C.2.4.1.19), for example, that harvested from cultures of strain A2-5a of the genus Bacillus.
Said Japanese Patent Kokai, however, discloses only the technical idea that solubilized hesperidin is admixed with intact hesperidin to prevent the precipitation of hesperidin crystals in products with hesperidin. Further, such a method gives a mixture of alpha-glucosyl hesperidin and hesperidin where intact hesperidin remains intact. Thus, canned products prepared by this method have the disadvantage that their syrups gradually become turbid. For coping with this problem, in the embodiments in the Japanese Patent Kokai, fractions of alpha-monoglucosyl and alpha-diglucosyl hesperidins are isolated, prior to uses. One may, however, encounter technical difficulties if he or she attempts to isolate the fractions at low cost.